PROJECT SUMMARY/ABSTRACT A perennial impediment in cellular biology has been the ability to design in vitro experiments that present a realistic representation of the in vivo environment. In order to reveal a particular cellular response or biochemical pathway requires that the cells in question reside in an appropriate and meaningful biological context. Unfortunately, a realistic biological environment always involves a complex milieu of neighboring cells that impose a unique physiochemical landscape with dynamic chemical, electrical and mechanical fields. This is especially true for embryological studies where a host of development morphogens establish complex chemical concentration profiles that drive cellular differentiation and tissue organization. Therefore, in order to effectively study detailed developmental mechanisms, experiments must be appropriately designed to duplicate these morphogen fields. This proposal presents the design, fabrication and characterization of a new microinstrument that generates controlled, user-defined concentration profiles that provide unprecedented control over the cellular environment for the study of chemical signaling mechanisms occurring during development. The microinstrument is used to generate morphogen fields that differentiate embryonic stem cells into organized neuromuscular tissue, complete with axon connections, and to study the mechanisms of mutual signaling between neuron and muscle cell that drive differentiation of specific neural subtypes. Results from this research will not only increase understanding of neuromuscular formation and neuro networks, but also impact limb and organ repair/regeneration as well as the diagnosis and treatment of degenerative neuromuscular diseases.